WO2018089374A1 - Compositions et méthodes pour inhiber la croissance de francisella - Google Patents

Compositions et méthodes pour inhiber la croissance de francisella Download PDF

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WO2018089374A1
WO2018089374A1 PCT/US2017/060435 US2017060435W WO2018089374A1 WO 2018089374 A1 WO2018089374 A1 WO 2018089374A1 US 2017060435 W US2017060435 W US 2017060435W WO 2018089374 A1 WO2018089374 A1 WO 2018089374A1
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host
day
francisella
antibiotic
treated
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PCT/US2017/060435
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Ky Van HOANG
John S. Gunn
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Ohio State Innovation Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Francisella tularensis subspecies tularensis (F. tularensis) is a remarkably infectious facultative intracellular bacterium, and the etiologic agent of tularemia, a zoonotic disease that affects a variety of mammals including humans.
  • F. tularensis infections can be acquired via aerosol, insect bites, or inoculation onto broken skin or mucous membranes [1].
  • F. tularensis can be divided into four subspecies including tularensis, novicida, holarctica, and mediasiatica [2, 3].
  • Tularemia is considered to be a re-emerging disease with recent outbreaks reported worldwide, including in the United States. Because of the ease of aerosol transmission, F. tularensis can be weaponized and could be deliberately transmitted, resulting in substantial morbidity and mortality on a large scale. It has therefore been recognized as a potential biological warfare agent and is classified as Tier 1, the highest-level bioterrorism agent classified by the U.S. Centers for Disease Control and Prevention (CDC) [4, 5].
  • F. tularensis can be difficult to treat because it is a facultative intracellular bacterium that targets macrophages and has several mechanisms that enable it to evade immune clearance [6].
  • LVS live attenuated vaccine strain
  • Antibiotic treatment of tularemia is limited primarily to aminoglycosides, fluoroquinolones (e.g. ciprofloxacin), and tetracyclines.
  • fluoroquinolones e.g. ciprofloxacin
  • tetracyclines e.g. ciprofloxacin
  • tigecycline ketolides, fluoroquinolones
  • improving antibiotic delivery in vivo e.g.
  • liposome delivery enhancement of the innate immune response by antimicrobial peptides, host-targeted therapy [13] and combinatorial approaches with conventional antibiotics and immune adjuvants [12,14].
  • AR-12 a small molecule derived from the COX- 2 inhibitor Celebrex, but lacking the COX-2 inhibitory activities, displayed broad- spectrum host-directed antimicrobial activity against fungi [15], Salmonella enterica serovar Typhimurium (S. Typhimurium) and . tularensis [13,16-18] wherein bacterial burdens were significantly reduced in the host macrophage, in part, through the induction of autophagy.
  • Several AR-12 derivatives exhibit direct antibacterial activities against methicillin-resistant Staphylococcus aureus [19], multidrug resistant tuberculosis [20], and Francisella (compound 20, herein called AR-16) [21].
  • AR-13 an AR-12 analog, has also been shown to have antibacterial activity, but not with respect to Francisella. See e.g.. U.S. Patent Application Publication 2013/0289004.
  • Francisella tularensis is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the U.S., and treatment of naturally acquired tularemia is confined to few antibiotics.
  • AR-13 exhibits direct in vitro bactericidal killing activity against Francisella, including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as towards the intracellular proliferation of LVS in macrophages, without causing appreciable toxicity to these host cells.
  • identification of an AR-13- resistant isolate indicates that this compound has an intracellular target(s), and that efflux pumps can mediate AR-13 resistance.
  • AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4.
  • Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death.
  • AR-13 has direct antimicrobial activities against Francisella species with a distinct mode of action compared with AR-16. While AR-13 displays bactericidal effects, AR-16 exhibits bacteriostatic activities against LVS and . tularensis SchuS4.
  • efflux pumps as an efflux pump inhibitor (e.g., carbonyl cyanide m-chlorophenyl hydrazone (CCCP), timcodar (also known as VX-853), ginsenoside 20(S)- Rh2, capsaicin, piperine, D-omithine-D-homophenylalanine-3-aminoquinoline (MC- 02,595, 2)) sensitized the AR-13 resistant mutant to AR-13.
  • an efflux pump inhibitor e.g., carbonyl cyanide m-chlorophenyl hydrazone (CCCP), timcodar (also known as VX-853), ginsenoside 20(S)- Rh2, capsaicin, piperine, D-omithine-D-homophenylalanine-3-aminoquinoline (MC- 02,595, 2)
  • aspects described herein provide methods of killing Francisella in a host infected with Francisella by administering AR-13 to the host to reduce the titer of Francisella by at least about 50 percent.
  • AR-13 and an antibiotic can be administered to the infected host.
  • the aminoglycosides can be selected from the group consisting of streptomycin, gentamicin, and amikacin.
  • the fluoroquinolone can be, for example, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifioxacin, sitafloxacin, and prulifloxacin.
  • the tetracycline is selected from the group consisting of doxycycline, minocycline, doxycycline, tetracycline, oxy tetracycline, tigecycline, chlortetracycline, lymecycline, meclocycline, methacycline, and rolitetracycline.
  • AR-13 is administered to the host in an amount sufficient to achieve a blood concentration of up to about 2.5 ⁇ g/ml.
  • the antibiotic can be administered to the host in an amount sufficient to achieve a therapeutic blood or tissue concentration (e.g., the amount provided in the product label for the antibiotic, product regulatory filings or literature).
  • FIG. 10 Further aspects provide methods of increasing the survival of a host infected with Francisella by administering AR-13 to the host wherein the survival of the host is increased by at least about 60% compared to an untreated host.
  • an antibiotic can also be administered to the host (e.g., aminoglycosides, fluoroquinolones, and tetracyclines).
  • Figure 1 provides the molecular structure of AR-12 and its derivatives AR-13 and AR-16;
  • Figure 2 A shows the effects of AR-13 and AR-16 on the optical densities at 600 nm (OD600) of LVS grown in 2-fold serial dilutions of AR-13 and AR-16 in mTSB as measured by a plate reader 18 h after inoculation;
  • Figure 2B shows the bactericidal effects of AR-13 on 1.5xl0 9 colony forming units (CFU) LVS incubated in 1 ml PBS (Phosphate Buffered Saline) containing 10 ⁇ g of AR-13;
  • Figure 2C shows the bacteriostatic effects of AR-16 on 1.5xl0 9 CFU LVS incubated in 1 ml PBS containing 10 ⁇ g of AR-16;
  • Figure 2D shows the bactericidal and bacteriostatic effects of AR-13 and AR- 16 on 1.5xl0 9 CFU of F. tularensis SchuS4 incubated in 1 ml PBS containing 10 ⁇ g of AR-13 or AR-16F;
  • Figure 2E shows exemplary effects of AR-13 on the growth of F. novicida in mTSB (modified tryptone soy broth) as measured by optical density (OD);
  • Figure 3 shows the intracellular growth of LVS in hMDMs following AR-13 treatment
  • Figure 4A shows exemplary growth curves of an LVS AR-13 resistant mutant (MT) compared to an LVS wild-type strain (WT) in the presence of various concentrations of AR-13 in mTSB;
  • Figure 4B illustrates the stability of AR-13 resistance in LVS MT strain by assessing viable bacteria four hours post treatment with AR-13;
  • Figure 5A shows that AR-13 resistant (AR-13 r ; MT) mutant to AR-13 is sensitized to AR-13 by adding a sub-inhibitory concentration of CCCP (4 nM);
  • Figure 5B shows that AR-13 resistance confers EtBr resistance in LVS. WT and MT were grown in 2-fold serial dilutions of EtBr;
  • Figure 5C shows that efflux pump inhibitor CCCP sensitizes the AR-13 r mutant to EtBr;
  • Figure 6A illustrates the protective effects of AR-13 in a mouse model of tularemia and provides exemplary survival curves of BALB/c mice infected by the intranasal (I.N.) route with 3xl0 3 CFU of LVS following AR-13 treatment;
  • Figure 6B provides exemplary survival curves of BALB/c mice infected by the I.N. route with 10 CFU of F. tularensis SchuS4 following AR-13 treatment;
  • Figure 6C provides exemplary survival curves of BALB/c mice infected I.N. with 10 CFU of F. tularensis SchuS4 following AR-13 treatment;
  • Figures 7A-7B shows the cytotoxicity of human monocyte-derived macrophages (hMDMs) cultured in the absence or presence of AR-13 or AR-16 for 24 hours ( Figure 7A) and 48 hours ( Figure 7B);
  • Figure 8 shows that the AR-13 resistant mutant does not confer resistance to kanamycin
  • Figure 9 provides an exemplary growth curve of AR-13 resistant mutant (MT) and wild-type LVS (WT) in different concentrations of efflux pump inhibitor CCCP in mTSB;
  • Figure 10A shows the exemplary effects of efflux pump proteins on AR-13 resistance in F. novicida (Fn);
  • Figure 10B shows the exemplary effects of transposon insertions in tolC on AR-13 resistance in Fn;
  • Figure IOC shows the exemplary effects of tolC homolog (ftn_0779) (ftl_l 107 homolog in LVS) on increasing susceptibility of AR-13 resistant mutants to AR-13;
  • Figure 10D shows the exemplary effects of a wbtH (ftl_0600 homolog in LVS) mutant on sensitivity of AR-13 resistant mutants to AR-13 ( Figure 10D).
  • compounds suitable for use alone or in combination with antibiotics as described herein include, for example, AR-13 (N- ⁇ 4-[5-(Phenanthren-2-yl)- 3-(trifluoromethyl)-lH-pyrazol-l-yl]phenyl ⁇ sulfuric diamide), having the following structure:
  • One aspect provides methods of killing Francisella in a host infected with Francisella, by administering AR-13 to the host, wherein the titer of Francisella in the host is reduced by at least about 50 percent compared to an untreated host.
  • the Francisella strain is selected from the group consisting of F. tularensis SchuS4 strain (Type A) and F. tularensis LVS.
  • the antibiotic is selected from the group consisting of aminoglycosides, fluoroquinolones, and tetracyclines.
  • the antibiotic is selected from the group consisting of streptomycin, gentamicin, and amikacin, doxycycline, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, sitafloxacin, and prulifloxacin.
  • the host can be treated with about 0.25 mg of gentamicin/kg/day.
  • AR-13 is administered to the host intranasally.
  • Gentamicin can be administered intravenously.
  • the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day.
  • the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day and 0.25 mg gentamicin/kg/day for at least about five consecutive days.
  • AR-13 is administered to the host in an amount sufficient to achieve a blood concentration of up to about 2.5 ⁇ g/ml.
  • Further aspects provide methods of increasing the survival of a host infected with Francisella, by administering AR-13 to the host wherein the survival of the host is increased by at least about 60% compared to an untreated host.
  • the Francisella strain is selected from the group consisting of F. tularensis SchuS4 strain (Type A) and . tularensis LVS.
  • This aspect also provid methods of administering AR-13 and an antibiotic to the infected host.
  • the antibiotic is selected from the group consisting of aminoglycosides, fluoroquinolones, and tetracyclines.
  • the antibiotic can be selected from the group consisting of streptomycin, gentamicin, and amikacin, doxycycline, ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, levofloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, sitafloxacin, and prulifloxacin.
  • the host can be treated with about 0.25 mg of gentamicin/kg/day.
  • AR-13 is administered to the host intranasally.
  • Gentamicin can be administered intravenously.
  • the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day.
  • the host is treated with from about 2.5 mg to about 5 mg AR- 13/kg/day and 0.25 mg gentamicin/kg/day for at least about five consecutive days.
  • AR-13 can be administered to the host in an amount sufficient to achieve a blood concentration of up to about 2.5 ⁇ g/ml.
  • the aspects described herein further comprise administering an efflux pump inhibitor to the host.
  • the efflux pump inhibitor can be selected from the group consisting of carbonyl cyanide m-chlorophenyl hydrazone, timcodar, ginsenoside 20(S)- Rh2, capsaicin, piperine, and D-ornithine-D-homophenylalanine-3-aminoquinoline.
  • AR-12 derivatives were screened to identify anti- Francisella agents using a standard serial dilution method, as previously higher concentrations of AR-12 were demonstrated to have adverse effects on the mammalian cells use in the in vitro studies.
  • Compounds AR-16 and AR-13 inhibit the growth of several Francisella subspecies with MICs of 2.5 ⁇ g/ml for F. tularensis LVS ( Figure 2A), and F. tularensis SchuS4 and 5 ⁇ g/ml for F. novicida ( Figure 2E) at 24-hour post- inoculation.
  • Figure 2A F. tularensis LVS
  • Figure 2E F. tularensis SchuS4 and 5 ⁇ g/ml for F. novicida
  • Only AR-13 had a bactericidal effect on Francisella.
  • An inhibitory effect of AR-13 has been observed in Mycobacterium tuberculosis [20], but not in serovars of the Gram-negative bacteria Salmonella (data not
  • AR-16 and AR-13 were used at a concentration of 10 ⁇ g/ml in PBS for bacterial killing assays. Viable bacteria were evaluated at different time points post- treatment by serial dilution, plating and enumeration. As discussed herein, AR-16 and AR- 13 have distinct modes of action: AR-13 has bactericidal activities and AR-16 has bacteriostatic effects on LVS and F. tularensis SchuS4 ( Figures 2B-2D), both at log and stationary phases. AR-13 treatment (10 ⁇ g/ml) leads to an approximate 2-3.5-log decrease in CFUs of F. tularensis SchuS4 and LVS, respectively, at 8 h post-treatment ( Figure 2B and D). Thus, AR-13 has significant bactericidal properties which can be used to control Francisella infection.
  • AR-13 induced minimal cytotoxicity towards hMDMs at AR-13 concentration as high as 40 ⁇ ( Figures 7A-7B). hMDMs were then infected with LVS and treated with different concentrations of AR-13. The intracellular bacterial load was evaluated 22 hours post-treatment. As shown in the Figure 3, AR-13 (10 ⁇ ) has inhibitory effects on the growth of LVS in hMDMs, reducing the CFU recovered by 0.5 logs. AR-12 was used as a control as it is known to inhibit intracellular Francisella growth by the induction of autophagy [17] and reduce LVS growth approximately 1 log at 5 ⁇ ( Figure 3). AR-12, however, is not known to have bactericidal effects on Francisella.
  • AR-13 resistant mutants were obtained. As shown in Figure 4A, a representative AR-13 resistant mutant was able to grow in the presence of 20 ⁇ AR-13. Examination of the stability of an AR-13 resistant mutant was performed by passing the mutant in AR-13- free mTSB for 10 overnight passes, with several clones then chosen for bacterial killing assays.
  • Glu441Lys were (Table 1).
  • the other mutation was in a gene encoding for a locus (FTL 0600) that is involved in O-antigen synthesis (amino acid substitution of
  • Table 1 Comparative genomic analysis of AR-13 resistant (AR-13 1 ) mutant and the wild-type (WT). Three non-synonymous mutations were identified in two putative efflux pumps (FTL l 107 and FTL 1865) in the genome of AR-13 r mutant.
  • the growth of the AR-13 resistant mutant was significantly decreased at 2.5 ⁇ AR-13 and nearly completely inhibited at 5 ⁇ AR-13 in the presence of CCCP while the strain grew normally at all concentrations of AR-13 (up to 10 ⁇ ) in the absence of a subinhibitory concentration of the inhibitor (Figure 5 A).
  • the AR-13 resistant isolate also conferred decreased sensitivity to ethidium bromide (EtBr) mediated by TolC as previously observed [27] ( Figure 5B), but does not affect sensitivity to kanamycin ( Figure 8). Similar to what was observed with AR-13, a sub-inhibitory concentration of CCCP sensitized the AR-13 resistant strain to EtBr ( Figure 5C).
  • Toxicity of AR-13 in mice was first evaluated in non-infected mice treated with 10 mg AR-13/kg/day intraperitoneally (IP) for 10 consecutive days. These mice did not show any abnormal clinical signs (sickness, hair ruffling) which indicated that they can tolerate at least total 100 mg AR-13/kg administered over a 10 day period. Mice were infected by the I.N. route with a lethal dose of LVS (3x10 3 CFUs/mouse) and then the infected mice were treated daily by I P. injection with 2.5 mg, 5 mg, or 10 mg AR-13/kg/day in 200 ⁇ PEG [13] for 10 days. A PEG-only treated group was included as a control.
  • AR-13 ability to control human virulent F. tularensis SchuS4 in the mouse infection model was examined. Without treatment, mice die from day 4-6 following intranasal (I.N.) infection with 10 CFU F. tularensis SchuS4 [13]. AR-13 was also tested in combination with a sub-optimal dose of gentamicin [13]. Mice were infected with 10 CFUs of F. tularensis SchuS4/mouse via the I.N. route and then treated with 2.5 mg or 5 mg AR-13/kg/day, or with 5 mg AR-13/kg/day plus 0.25 mg gentamicin/kg/day (LP.) for five consecutive days.
  • AR-13 or the compositions described herein can be used to treat or prevent the illness and/or disease caused by infectious microbes including, but not limited to, those listed above.
  • AR-13 can be provided to an infected patient concurrently with an antibiotic or serially in any suitable order.
  • AR-13 and the antibiotic can be administered to patients simultaneously or AR-13 and the antibiotic can be formulated together.
  • AR-13 can be formulated together with an efflux pump inhibitor as described herein.
  • AR-13, an antibiotic, and an efflux pump inhibitor can be formulated together.
  • AR-13 directly kills different Francisella subspecies including F. tularensis SchuS4, LVS ( Figure 2), and F. novicida ( Figure 2E).
  • "directly kills” means killing 99.9% of a bacterial inoculum within a 24-hour exposure period ([29]) of AR-13.
  • a time-kill kinetic effect on LVS is approximately 3 and 4 log CFU reduction at 8 hours post treatment for F. tularensis SchuS4 and LVS, respectively (See, e.g.. Figures 2B and D.
  • AR-13 demonstrates in vivo mti-Francisella activity. Despite infection with a lethal dose of LVS, two of four mice treated with 2.5 mg/kg/day (total 25 mg/kg for the whole course of treatment) of AR-13 recovered and survived to the study endpoint, while none of the vehicle-treated control mice survived.
  • administer refers to providing the compositions described herein to a patient including by the patient, a healthcare professional, a caretaker, and also includes prescribing the compositions described herein to the patient.
  • compositions described herein can be administered orally, parenterally (intravenously [IV], intramuscularly [IM], depot-IM, subcutaneously [SQ], and depot-SQ), sublingually, intranasally, by inhalation, intrathecally, topically, or rectally.
  • IV intravenously
  • IM intramuscularly
  • SQ subcutaneously
  • SQ subcutaneously
  • depot-SQ sublingually
  • intranasally by inhalation
  • intrathecally topically, or rectally.
  • AR-13 can be formulated into suitable pharmaceutical preparations such as creams and gels, for topical application; suspensions, tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration, suspensions or solutions appropriate for inhalation (e.g., metered dose inhalers, dry powder inhaler, nanoparticles) and lyophilized formulations for parenteral administration.
  • suitable pharmaceutical preparations such as creams and gels, for topical application; suspensions, tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration, suspensions or solutions appropriate for inhalation (e.g., metered dose inhalers, dry powder inhaler, nanoparticles) and lyophilized formulations for parenteral administration.
  • AR-13 can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.
  • about 0.1 to 1000 mg, about 5 to about 200 mg, or about 10 to about 50 mg of the AR-13, or a physiologically acceptable salt or ester can be
  • compositions or preparations comprising AR-13 are compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
  • a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc. in a unit dosage form as called for by accepted pharmaceutical practice.
  • the amount of active substance in compositions or preparations comprising AR-13 is such that a suitable dosage achieving the therapeutic range indicated is obtained.
  • compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 1000 mg, about 1 to about 500 mg, or about 10 to about 200 mg of the active ingredient.
  • unit dosage from refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • one or more of AR-13 is mixed with a suitable pharmaceutically acceptable carrier to form compositions.
  • a suitable pharmaceutically acceptable carrier to form compositions.
  • the resulting mixture may be a cream, gel, solution, suspension, emulsion, or the like.
  • Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. In one aspect, the effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.
  • compositions or vehicles suitable for administration of AR-13 described herein include any such carriers suitable for the particular mode of
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • methods for solubilizing may be used. Such methods are known and include, but are not limited to, using co-solvents such as ethanol (EtOH) or dimethylsulfoxide (DMSO), using surfactants (e.g., anionic, cationic, zwitterionic, and non-ionic). Specific suitable surfactants include, but are not limited to, TWEEN, poloxamer, sodium lauryl sulfate, aluminum monostearate and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs, may also be used in formulating effective pharmaceutical compositions.
  • co-solvents such as ethanol (EtOH) or dimethylsulfoxide (DMSO)
  • surfactants e.g., anionic, cationic, zwitterionic, and non-ionic.
  • surfactants include, but are not limited to, TWEEN, poloxamer, sodium lauryl sulfate, aluminum monostearate and dissolution
  • the concentration of the compound is effective for delivery of an amount upon administration that lessens or ameliorates at least one symptom of the disorder for which the compound is administered.
  • the compositions are formulated for single dosage administration.
  • AR-13 as described herein may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings.
  • Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems.
  • the active compound can be included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective dose may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder.
  • Such carriers include controlled release formulations, such as, but not limited to, implants and
  • microencapsulated delivery systems and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polygly colic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.
  • AR-13 and compositions described herein can be enclosed in multiple or single dose containers.
  • the enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use.
  • AR-13 in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use.
  • a kit may include AR-13 and a second therapeutic agent for co-administration.
  • AR-13 and second therapeutic agent may be provided as separate component parts.
  • a kit may include a plurality of containers, each container holding one or more unit dose of AR-13 described herein.
  • the containers can be adapted for the desired mode of administration, including, but not limited to suspensions, tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, gels, suspensions, creams, and the like for topical administration.
  • concentration of AR-13 in the pharmaceutical composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches.
  • Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient (e.g., any suitable filler/bulking agent) such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.
  • a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin
  • an excipient e.g., any suitable filler/bulking agent
  • the dosage unit form when it is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.
  • the active materials can also be mixed or co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action.
  • AR-13 can be used, for example, in combination with an antibiotic, antiviral, antifungal, or pain reliever.
  • solutions or suspensions used for parenteral, intradermal, subcutaneous, inhalation, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, alcohols, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA);
  • a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as
  • buffers such as acetates, citrates, and phosphates
  • agents for the adjustment of tonicity such as sodium chloride and dextrose.
  • Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.
  • suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, ethanol, N-methylpyrrolidone, surfactants and mixtures thereof.
  • PBS phosphate buffered saline
  • suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, ethanol, N-methylpyrrolidone, surfactants and mixtures thereof.
  • Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known in the art.
  • compounds employed in the methods of the disclosure may be administered enterally or parenterally.
  • compounds employed in the methods of the disclosure can be administered in usual dosage forms for oral administration as is well known to those skilled in the art.
  • These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs.
  • the solid dosage forms can be of the sustained release type so that the compounds employed in the methods described herein need to be administered only once or twice daily.
  • terapéuticaally effective amount and “therapeutically effective period of time” are used to denote treatments at dosages and for periods of time effective to reduce microbial burden.
  • administration can be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal.
  • the therapeutic composition can be administered at a sufficient dosage to attain a blood or tissue level of the compounds of from about 0.1 ⁇ to about 20 ⁇ .
  • a blood or tissue level of the compounds of from about 0.1 ⁇ to about 20 ⁇ .
  • concentrations for localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated.
  • One skilled in the art will appreciate that such therapeutic effect resulting in a lower effective concentration of AR- 13 may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated. It is also understood that while a patient may be started at one dose, that dose may be varied overtime as the patient's condition changes.
  • F. novicida mutant strains were obtained from BEI Resources transposon library (https://www.beiresources.org). The bacteria were cultured on chocolate II agar (CHA) plates (Becton Dickinson, Sparks, MD) or modified Tryptic Soy Broth (mTSB) or agar [24] for 48 h (F. tularensis SchuS4 and LVS) or for 24 h (F. novicida) at 37 ° C prior to use in all experiments. Experiments involving the LVS strain were performed in a BLS2 environment.
  • MICs minimum inhibitory concentrations
  • bacterial killing assays were performed by a standard microtiter broth dilution method with an inoculum of approximately lxlO 6 bacteria ml as described previously [24, 25]. MICs were determined by the lowest concentration of specific antimicrobial showing complete inhibition of bacterial growth after 24 h of incubation at 37°C.
  • bacterial killing assays bacterial strains were grown at 37 ° C in mTSB or agar by supplementing with 135 ⁇ g/ml ferric pyrophosphate and 0.1% cysteine hydrochloride at 37 ° C for 48 hours.
  • PBS phosphate buffered saline
  • OD optical density
  • AR-13 was used as the selective agent to obtain spontaneous AR-13 -resistant (AR-13 1 ) mutants in vitro by stepwise selection in broth culture. 50 ⁇ from an overnight culture of LVS was exposed to increasing concentrations of AR-13 in 5 ml mTSB broth with 0.25 ⁇ g AR-13/ml as a starting concentration at 37°C while shaking. 50 ⁇ of this LVS grown culture was then passed into 2-fold increasing concentrations of AR-13 in 5 ml mTSB. The process was repeated until LVS was able to stably grow in 20 ⁇ g AR-13/ml (approximately 20 passes). Approximately 70 AR-13 r clones were selected to determine the MIC to AR-13. Two representative AR-13 r mutants were then passed in non-selective AR-13 free mTSB for 10 passes of an overnight culture. The stable AR-13 r mutants were chosen for the subsequent studies including genomic sequencing and assays regarding the mechanism of AR-13 resistance.
  • hMDMs Human monocyte-derived macrophages
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs were then cultured in sterile screw-cap Teflon wells in RPMI 1640 plus L- glutamine (Gibco-Life Technologies, Grand Island, NY) with 20% autologous human serum at 37°C in a humidified incubator containing 5% C02 for 5 days.
  • PBMCs were then recovered from Teflon wells by chilling them on ice, re-suspending the cells in RPMI 1640 with 10% autologous serum, and allowing them to attach in 6-well or 24-well tissue culture plates for 2-3 h at 37°C in a humidified incubator containing 5% C02.
  • Lymphocytes were then washed away leaving the hMDM monolayers at a density of approximately 2.0x l0 5 cells/well for 24-well plates for LVS infection.
  • LVS LVS were grown at 37°C in mTSB or agar by supplementing with 135 ug/ml ferric pyrophosphate and 0.1% cysteine hydrochloride at 37°C for 48h. Bacteria were suspended in PBS to an OD of 0.4 at 600nm, equivalent to 3x10 9 CFU/ml. hMDMs were infected with LVS at a multiplicity of infection (MOI) of 50 in the presence of 2% autologous serum in RPMI 1640 plus L-glutamine (Gibco-Life Technologies).
  • MOI multiplicity of infection
  • AR-12 and AR-13 were dissolved at a concentration of lOmg/mL in DMSO and diluted in RPMI 1640 containing 2% autologous serum to the appropriate concentrations.
  • the infected cells were lysed with 0.1% Triton X-100 (Calbiochem, San Diego, CA) in PBS for 15 min.
  • the cell lysates were then serially diluted with PBS and spread on CHA plates.
  • the level of surviving intracellular bacteria was determined by enumerating CFU after 72 hours incubation at 37°C.
  • Intranasal (I.N.) infection of F. tularensis SchuS4 and LVS was performed as previously described [13]. Briefly, F. tularensis SchuS4 and LVS were grown on CHA plates for 48 h at 37oC. The bacteria were collected, suspended in PBS, and adjusted to an OD of 0.4 at 600nm, equivalent to 3x10 9 CFU/ml. The desired concentrations of F.
  • mice were infected with 10 CFU of F. tularensis SchuS4 in 50 ⁇ PBS.
  • mice were infected by the I.N. route with 3x10 3 CFU in 50 ⁇ 1 PBS.
  • mice Prior to the infection, mice were anesthetized with isoflourane as approved by The Ohio State University Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • I P. intraperitoneal
  • the infected mice were monitored for survival up to two weeks post-infection.
  • 5 mice/group were infected with the LVS strain via the I.N. route and treated daily with 5 mg AR-13 in 200 ⁇ PEG/kg/day from day 0.
  • bacterial burdens in the lung of infected mice were determined by tissue homogenization and plating for CFU enumeration.
  • Example 10 [000109] Data are presented as mean ⁇ standard deviation (SD). P-values were calculated using one-way ANOVA for multiple comparisons and adjusted with

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Abstract

L'invention concerne des compositions et des méthodes de prévention et de traitement d'un hôte infecté par Francisella par traitement de l'hôte avec AR-13 seul ou en combinaison avec des antibiotiques.
PCT/US2017/060435 2016-11-08 2017-11-07 Compositions et méthodes pour inhiber la croissance de francisella WO2018089374A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110218239A1 (en) * 2006-11-09 2011-09-08 University Of Maryland Baltimore Use of 5,6-Dimethylxanthenone-4-Acetic Acid as an Antiviral Agent
US20130237575A1 (en) * 2007-07-24 2013-09-12 The Ohio State University Research Foundation Anti-infective agents against intracellular pathogens
US20150258100A1 (en) * 2012-07-30 2015-09-17 The Ohio State University Antibacterial protein kinase inhibitors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110218239A1 (en) * 2006-11-09 2011-09-08 University Of Maryland Baltimore Use of 5,6-Dimethylxanthenone-4-Acetic Acid as an Antiviral Agent
US20130237575A1 (en) * 2007-07-24 2013-09-12 The Ohio State University Research Foundation Anti-infective agents against intracellular pathogens
US20150258100A1 (en) * 2012-07-30 2015-09-17 The Ohio State University Antibacterial protein kinase inhibitors

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